Luminescence Reference Standards

ABSTRACT

The present teachings provide for systems, and components thereof, for detecting and/or analyzing light. These systems can include, among others, optical reference standards utilizing luminophores, such as nanocrystals, for calibrating, validating, and/or monitoring light-detection systems, before, during, and/or after sample analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.12/338,778 filed Dec. 18, 2008; which is a continuation of patentapplication Ser. No. 11/173,609 filed Jun. 30, 2005, now U.S. Pat. No.7,480,042; which claims a priority benefit under 35 U.S.C. §119(e) fromU.S. Patent Application No. 60/584,890 filed Jun. 30, 2004; all of whichare incorporated herein by reference.

INTRODUCTION

The identity, properties, and interactions of samples such asbiomolecules can be analyzed by detecting light emitted by or otherwiseoriginating from the sample. The emitted light can arise naturally inthe sample and/or be induced by illumination or other stimulus. Ineither case, the emitted light can be detected, and the illuminationoptionally can be provided, by a suitable light-detection system. Theability of the light-detection system to collect and quantify emittedlight and thus to characterize samples is determined in part by theextent to which the light-detection system is and can be calibrated.

SUMMARY

The present teachings provide systems, and components thereof, fordetecting and/or analyzing light. These systems can include, amongothers, optical reference standards for calibrating, validating, and/ormonitoring light-detection systems, before, during, and/or after sampleanalysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary light-detection system,showing selected optical components and an optical reference standard,in accordance with aspects of the present teachings.

FIG. 2 is a top view of an exemplary optical reference standard, inaccordance with aspects of the present teachings.

FIG. 3 is an exploded isometric view of two exemplary embodiments of anoptical reference standard, with an upper light-blocking portion and alower light-producing portion, in accordance with aspects of the presentteachings. The embodiment shown at left is configured generally for usein an optical plane intermediate between a light source and the objectplane. The embodiment shown at right is configured generally for use inthe object plane.

FIG. 4 is an exploded isometric view of two alternative exemplaryembodiments of an optical reference standard, with an upperlight-producing portion and a lower light-blocking portion, inaccordance with aspects of the present teachings. The embodiment shownat left is configured generally for use in an optical plane intermediatebetween a light source and the object plane. The embodiment shown atright is configured generally for use in the object plane.

FIG. 5 is a schematic sectional view of an exemplary subtraction-basedoptical reference standard, showing light-producing and light-blockingelements, in accordance with aspects of the present teachings.

FIG. 6 is an exemplary emission spectrum for Ultem® 1000 plastic,showing relative emission intensity as a function of emissionwavelength, following excitation at a fixed excitation wavelength, inaccordance with aspects of the present teachings. The shaded envelope isconstructed from the lowest and highest intensities measured at eachemission wavelength, based on analysis of 384 identically preparedsamples.

FIG. 7 is a plot of transmission versus wavelength showing individualand composite transmission spectra of subtractive elements of thesubtraction-based, Ultem®-1000-plastic-containing reference standard ofFIG. 6. The dashed, dot-dashed, and thin solid lines representtransmission spectra of individual layers. The thick solid linerepresents the composite transmission spectrum.

FIG. 8 is a plot of an exemplary calculated emission spectrum for thesubtraction-based, Ultem®-1000-plastic-containing reference standard ofFIG. 6. The calculated emission spectrum (solid line) is a product ofthe emission spectrum of a broadband photoluminescent component (thickdashed line) and the composite transmissivity of various spectral filtercomponents (thin dashed line).

FIG. 9 is an exemplary emission spectrum for a subtraction-based,Ultem®-1000-plastic-containing reference standard, showing relativeemission intensity as a function of emission wavelength, followingexcitation at a fixed excitation wavelength, in accordance with aspectsof the present teachings. The shaded envelope is constructed from thelowest and highest intensities measured at each emission wavelength,based on analysis of 384 identically prepared samples.

FIG. 10 is an exemplary emission spectrum for an addition-based,photoluminescent-nanocrystal-containing reference standard, showingrelative emission intensity as a function of emission wavelength,following excitation at a fixed excitation wavelength, in accordancewith aspects of the present teachings. The shaded envelope isconstructed from the lowest and highest intensities measured at eachemission wavelength, based on analysis of 20 identically preparedsamples.

FIG. 11 is an exemplary emission spectrum for an addition-based,luminescent-glass-containing reference standard, showing relativeintensity as a function of emission wavelength, following excitation ata fixed excitation wavelength, in accordance with aspects of the presentteachings. The shaded envelope is constructed from the lowest andhighest intensities measured at each emission wavelength, based onanalysis of a plurality of identically prepared samples.

FIG. 12 is a schematic sectional view of another exemplarylight-detection system, showing selected optical components and anoptical reference standard, in accordance with aspects of the presentteachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

The present teachings provide systems, including components thereof andaids thereto, for detecting and/or analyzing light. These systems caninclude, among others, optical reference standards for calibrating,validating, and/or monitoring light-detection systems, includingcomponents and/or aspects thereof, before, during, and/or after sampleanalysis. Suitable uses can include aligning portions of a lightdetection system, relative to one another and/or one or more samples.Suitable uses also can include validating system performance, includingattainment and/or maintenance of threshold conditions. Suitable usesalso can include monitoring and/or correcting for underages, overages,and/or variations in illumination intensity, among others. Theseunderages, overages, and/or variations can occur with respect to spaceand/or time, and can be determined and/or corrected for before, during,and/or after sample analysis. Thus, the optical reference standards canbe used in methods of calibrating, validating, and/or monitoring alight-detection system and/or methods of performing an assay (e.g.,involving measuring properties of the reference standard and sample, andperforming a correlation and/or correction based on such measurements),among others.

Exemplary usages of the optical reference standards can include (1)aligning optical components, (2) validating system performance, (3)confirming performance and settings for excitation and/or emissionfilters (before or after their installation in the same or a differentinstrument), (4) verifying threshold illumination (e.g., intensityand/or uniformity) and detection (e.g., sensitivity) parameters, (5)compensating for spatial and/or temporal variations in illumination, (6)tracking drift in illumination intensity and/or looking for signs of(and acting to prevent) impending component (e.g., light source)failure, (7) locating and/or aligning sample holders, or portionsthereof (e.g., in high-density array grids), and/or (8) calibratingintensity, wavelength, and/or other parameters (e.g., by comparingvalues measured in a sample against known values measured from thestandard), among others. Additional usages and applications aredescribed below, particularly in Section III and Example 4, amongothers.

Exemplary users can include manufacturers, service and maintenancepersonnel, and/or end users, among others.

FIG. 1 shows an exemplary light-detection system 100, including selectedoptical components 102-108 and an optical reference standard 110, inaccordance with aspects of the present teachings.

The light-detection system generally comprises any mechanism fordetecting (and optionally analyzing) light from a sample. Suitablesystems can include (1) a light source 102 for producing light forilluminating, and/or inducing a suitable or desired response from, asample, (2) a sample-support device 104 for supporting one or moresamples 112 at an examination area 114, (3) a detector 106 for detectinglight transmitted or otherwise originating from a sample and optionallyconverting the detected light into a representative signal, and/or (4)an optical relay structure 108 for directing light between the lightsource and examination area, and the examination area and detector,among others. These and other aspects of the light-detection system aredescribed below; see particularly Section II and Example 3. Exemplarylight-detection systems, including components and uses thereof, also aredescribed in the following patent application, which is incorporatedherein by reference: U.S. Provisional Patent Application Ser. No.60/584,525, filed Jun. 30, 2004, titled Distributed Light Detector ofinventors Charles S. Vann and Steven Boege.

The optical reference standard generally comprises any mechanism formonitoring and/or calibrating a light-detection system, includingcomponents and/or aspects thereof. The reference standard can include alight-producing (e.g., luminescent) portion 116 and/or a light-blocking(e.g., mask) portion 118, among others. The reference standard can bedisposed in the light-detection system, transiently or permanently, atany suitable position(s). Typical positions lie in or along the opticalpath 120, such as in the object plane 122 (i.e., the plane, typically atleast partially coextensive with the examination area, that is occupiedby the sample and imaged by the detector during sample analysis) and/orin an intermediate position 124 (e.g., an intermediate image plane)operatively disposed along the incident illumination path between thelight source and object plane, among others. The reference standard canbe positioned manually and/or automatically, as desired. The referencestandard can be placed into and/or taken out of the light path inconnection with each use and/or related series of uses, and/or thereference standard can be positioned permanently in the light path. Thereference standard (or a suitable set of reference standards) can behoused within the detection system, where it can be moved more easilyinto or out of position or, alternatively, maintained in a fixedposition. These and other aspects of the optical reference standard aredescribed below; see particularly Section I and Examples 1 and 2.

The following sections describe further aspects of the presentteachings, including (I) reference standards, (II) light-detectionsystems, (III) applications, and (IV) examples, among others.

I. OPTICAL REFERENCE STANDARDS

The optical reference standard, as noted above, generally comprises anymechanism for monitoring and/or calibrating a light-detection system,including components and/or aspects thereof.

FIG. 2 shows an exemplary optical reference standard 200, in accordancewith aspects of the present teachings. This reference standard caninclude a light-producing (e.g., luminescent) element 202, for producinglight, and/or a light-blocking (e.g., mask) element 204, for selectivelyblocking light, among others. These elements can be present and/oremployed singly and/or in combination. Thus, in a given light-detectionsystem, and/or for a given application, a light-producing element can beused, a light-blocking element can be used, and/or both alight-producing and a light-blocking element can be used. Moreover, ifused together, the light-producing and light-blocking elements can be atleast substantially separate or at least substantially unitary,depending on embodiment. Thus, in some cases (e.g., FIG. 3), thelight-blocking element can be supported by or ancillary to thelight-producing element, whereas, in other cases (e.g., FIG. 4), thelight-producing element can be supported by or ancillary to thelight-blocking element. In both cases, the light-producing andlight-blocking elements simply can be placed in contact or proximity, orthe light-producing and light-blocking elements can be joined and/orrendered at least partially coextensive using any suitable mechanism(including adhesives, fasteners, fusion, and/or so on).

The light-producing and light-blocking elements (as well as othercomponents of the reference standard) can have any suitable shape orsize. Commonly, these elements, or portions thereof, will be at leastsubstantially planar. Such planar portions can be configured so that, inuse, the plane is at least substantially perpendicular to the opticalaxis of the light-detection system. More generally, the referencestandard (or components thereof) can be shaped and/or sized to match astandard sample holder, or portion thereof, such as a microplate, abiochip, and so on.

The properties of the optical reference standard can be correlated withproperties of the intended sample(s). The correlation can includematching properties of the light produced by the standard withproperties of the light produced by the sample(s), including wavelength,intensity, polarization, and/or the like. The correlation also caninclude matching the concentration, size, and/or position ofluminophore(s) in the reference standard and sample(s).

The reference standard can include one or more ancillary elements,helpful to function, but not involved directly in light production.These ancillary elements can include an identification element 206,among others. The identification element can include any mechanism orfeature for identifying the reference standard and/or a characteristicor use thereof. Suitable mechanisms or features can include a barcodeand/or a shape, pattern, marking, aperture, or the like associated withthe reference standard. Suitable information can include a uniqueidentifier (e.g., a serial number), general properties of the referencestandard (e.g., emission wavelength profile, the identities ofluminophores suitable for use with the standard, assay conditions oridentifiers, operator information, date, etc.), and/or so on. Theinformation provided by the identification element can be used for anysuitable purpose, including setting detection-system parameters (e.g.,filter settings), inputting data to be stored electronically with theassay results, and/or so on.

The reference standard can be configured for use in any desired opticalconfiguration, including trans (excitation light directed to andemission light collected from opposite sides of the sample), epi(excitation light directed to and emission light collected from sameside of the sample), and oblique (excitation light directed and emissionlight detected at oblique angles), among others. For example, for transillumination, associated support components typically would betransparent to at least one of the excitation and emission light,whereas for epi illumination such supports can be transparent or opaque.

The reference standard can be placed in and/or removed from desiredpositions in the light-detection system by any suitable mechanism. Forexample, the reference standard can be placed in the examination area byhand, and/or carried to and/or from the examination area (or otherposition) by an automated conveyor mechanism, among others. The conveyormechanism can be the same as or different than any mechanism (such as astage) used to convey sample holders to and/or from the examinationarea. Where the same conveyor is used for both standards and samples,the conveyor can be adapted to hold standards and samples simultaneouslyand/or sequentially. Where different conveyors are used for standardsand samples, the two conveyors can be configured and controlledindependently and/or coordinately. In either case, the standardoptionally can be configured (e.g., shaped and/or sized) like the sampleholder, simplifying handling, and helping to ensure that the standardsuccessfully emulates the sample, sample conditions, and/or aspectsthereof.

These and other aspects of optical reference standards are describedbelow, including (A) relationships between light-producing andlight-blocking elements, (B) light-producing elements, and (C)light-blocking elements, among others. Attributes of these elements, andcomponents thereof, can be mixed and matched as appropriate or desired.

I.A Relationships Between Light-Producing and Light-Blocking Elements

This section describes exemplary relationship between thelight-producing and light-blocking elements, in the context of severalexamples; see FIGS. 3 and 4.

I.A(i) Exemplary Relationships 1

FIG. 3 shows two exemplary embodiments of a first optical referencestandard 300, with a first set of relationships between light-producingand light-blocking elements, in accordance with aspects of the presentteachings. These reference standards are particularly suitable for usein epi illumination.

Reference standard 300 can include a light-producing element 302 a,b(corresponding to embodiments “a” and “b”) and a light-blocking element304, among others. Light-producing element 302 a in embodiment “a” caninclude a luminescent portion 306 a and a set of transparent portions308. Light-producing portion 302 b in embodiment “b” can include aluminescent portion 306 b but does not (necessarily) include anytransparent portions. Light-blocking element 304 (in both embodiments)can include an opaque portion 310 and first and second sets 312, 314 oftransparent portions, respectively. The first set of transparentportions is configured for sample excitation; specifically, in thepictured embodiment, there are 96 apertures configured to allow light totravel to and from 96 samples in a 96-well microplate. The second set oftransparent portions is configured for calibration; specifically, in thepictured embodiment, there are 8 inter-well apertures configured toallow light to travel to and from an associated light-producing element.Here, transparent portions can transmit at least a substantial portionof incident light, and opaque portions can block at least substantiallyall incident light.

Embodiment “a” (i.e., the left side of the reference standard (302a+304)) is configured for placement in an intermediate optical plane,between the light source and sample, facilitating use before, during,and/or after sample analysis. In use, the first set of transparentportions (312) in the light-blocking element can be aligned with thetransparent portions (308) in the light-producing element, allowingexcitation light, at first preselected positions, to impinge on samplesand induce production of emission light that passes back through thealigned sets of transparent portions and onto the detector, where it canbe used for sample analysis. Moreover, the second set of transparentportions (314) in the light-blocking element can be aligned withemissive portions (306 a) of the light-producing element, allowingexcitation light, at second preselected positions, to impinge on anadjacent light-producing element and induce production of emission lightthat passes back through the first set of transparent portions and ontothe detector, where it can be used for calibration. These concepts arefurther illustrated in FIG. 1.

Embodiment “b” (i.e., the right side of the reference standard (302b+304)) is configured for placement in an object plane, facilitating usebefore and/or after sample analysis. Here, in use, both the first (312)and second (314) sets of transparent portions in the light-blockingportion can be aligned with emissive portions (306 b) of thelight-producing element, allowing excitation light to impinge on andinduce production of emission light from both regions, where it can beused for calibration.

I.A(i) Exemplary Relationships 2

FIG. 4 shows two exemplary embodiments of a second optical referencestandard 400, with a second set of relationships between light-producingand light-blocking elements, in accordance with aspects of theinvention. These reference standards, like those in FIG. 3, areparticularly suitable for use in epi illumination.

Reference standard 400 can include a light-producing element 402 a,b(corresponding to embodiments “a” and “b”) and a light-blocking element404 a,b, among others. Light-producing element 402 a in embodiment “a”can include a set of light-emissive portions 406, and light-producingelement 402 b in embodiment “b” can include first and second sets 408,410 of light-emissive portions. Light-blocking element 404 a can includean opaque portion 412 a and a set 414 of transparent portions.Light-blocking element 404 b can include an opaque portion 412 b butdoes not (necessarily) include any transparent portions. The picturedembodiment is configured for use with a 96-well microplate, in parallelwith FIG. 3, although, like in FIG. 3, the general principles can beapplied in any suitable manner. Here, the light-emissive portions (406,408, and 410) are supported by (i.e., contact, rest upon, and/or arepartially or completely embedded in) the light-blocking element (404 aor 404 b, respectively). The light-producing portions (in this and/orother embodiments) can independently have the same and/or differentspectral outputs (i.e., can independently produce light with the sameand/or different color(s)).

Embodiment “a” (i.e., the left side of the reference standard (402 a+404a)) is configured for placement in an intermediate optical plane,between the light source and sample. In use, light can travel to andfrom the sample through transparent portions 414, for use in sampleanalysis, and light emission can be induced from light-emissive portions406, for use in calibration. This embodiment most closely resemblesembodiment “a” in FIG. 3.

Embodiment “b” (i.e., the right side of the reference standard (402b+404 b)) is configured for placement in an object plane. Here, in use,light emission can be induced from both the first (408) and second (410)sets of light-emissive portions, for use in calibration. This embodimentmost closely resembles embodiment “b” in FIG. 3.

I.B Light-Producing Elements

The light-producing element generally comprises any mechanism forproducing (and/or redirecting) light as part of an optical referencestandard for monitoring and/or calibrating a light-detection system.This element can be distinguished by the nature and/or arrangement ofthe mechanism(s) used to produce the light and/or the disposition of themechanism(s) relative to an associated light-blocking element and/or alight-detection system, among others. Thus, in some embodiments, andunless otherwise noted, the light-producing element can be configured toproduce light by any suitable mechanism, including emission, reflection,transmission, refraction, diffraction, and/or scattering, among others.Here, emission can include luminescence, including photoluminescence(e.g., fluorescence, phosphorescence, etc.) and/or chemiluminescence,among others. Conversely, in other embodiments, the light-producingelement can be configured to produce light only via particularmechanisms, for example, via luminescence mechanisms, as described belowfor subtraction-based and addition-based reference standards. In eithercase, the light produced by the light-producing element can be used “asis” and/or filtered to alter its wavelength, intensity, polarization,and/or coherence, among others.

The optical reference standard can be configured to produce lightprimarily or exclusively at preselected wavelengths and/or overpreselected ranges of wavelengths. Toward this end, in some“subtraction-based” embodiments, the light produced by the referencestandard can be obtained by “subtracting” undesired spectral componentsfrom a relatively broadband emission; see Section I.D below. In other“addition-based” embodiments, the light produced by the referencestandard can be obtained by “adding” desired spectral components fromtwo or more materials; see Section I.E below. In yet other embodiments,the light produced by the reference standard can be obtained by acombination of these and/or other mechanisms.

Luminescence-based light-producing elements can be based onphotoluminescence, in which the light-producing element is induced toemit emission light by illumination with suitable excitation light. Theemission light corresponding to excitation with particular excitationlight can be characterized by an emission spectrum, and the excitationlight corresponding to particular emission light can be characterized byan excitation spectrum, as described below. Typically, the excitationlight will lie at least primarily to one side of some cutoff wavelength,and the emission light will lie at least primarily to the other side ofthe cutoff wavelength. For example, with single-photon excitation, thewavelength(s) of the excitation light typically would be shorter thansome cutoff wavelength (corresponding to relatively higher energies),and the wavelength(s) of the emission light typically would be longerthan the cutoff wavelength (corresponding to relatively lower energies).Conversely, with multi-photon excitation, these wavelength differencestypically would be reversed. The differences between the wavelengths ofthe excitation and emission light can facilitate the separation anddistinguishable detection of emission light.

I.C Light-Blocking Elements

The light-blocking element generally comprises any mechanism forblocking (and/or redirecting) light as part of an optical referencestandard for monitoring and/or calibrating a light-detection system.This element can be distinguished by the nature and/or arrangement ofthe mechanism(s) used to block the light and/or the disposition of themechanism(s) relative to an associated light-producing element and/or alight-detection system, among others. The light-blocking (or opticallyopaque) element can be configured to block light by any suitablemechanism, including absorption and/or reflection, refraction,diffraction, and/or scattering out of the optical path, among others.The blocked light can represent any amount between at least about halfand at least about all (or entirely all) of the incident light, amongothers.

The light-blocking element can be formed of any suitable material,including but not limited to plastic and/or metal such as aluminum orsteel (e.g., sheet metal) having a non-fluorescing coating or paint,such as black anodizing.

The light-blocking element can include both light-blocking (opaque)portions, as described above, and light-transmitting (transparent)portions that allow passage of excitation and/or emission light, asappropriate or desired. The opacity and transparency of these portionscan be absolute or relative. Collectively, the opaque and transparentportions can create a mask or pattern that allows selective illuminationof the light-producing portion of the reference standard and/or samplesin a light-detection system, for example, as described above for FIGS.1, 3, and 4.

The transparent portions (and thus the opaque portions) can have anysuitable shape and size. For example, the transparent portions can besized and spaced to correspond to and/or allow selective illumination ofa light-producing element and/or sample holder, among others. Thus, inspecific embodiments, the reference standard can include 96, 384, or1536 transparent portions, among others, corresponding to wells in a 96,384, or 1536-well microplate, among others. Alternatively, or inaddition, the reference standard might include another number oftransparent portions corresponding to inter-well calibration marks.

The transparent portions can have any form capable of transmittingexcitation and/or emission light, or portions thereof. Thus, thetransparent portions can include apertures and/or optically transparent(or partially transparent) materials (at least in the wavelengthrange(s) of interest). The optically transparent materials can includeglass, quartz, plastic and/or the like, among others.

I.D Subtraction-Based Reference Standards

Subtraction-based reference standards generally comprise any referencestandards, or portions thereof, in which output light with the desiredspectral components is obtained by producing light with both desired andundesired spectral components and then “subtracting” the light with theundesired spectral components. The subtraction-based reference standardscan include, among others, (1) a relatively broadband emissive element,and (2) a relatively narrowband subtractive element. The emissiveelement generally is capable of outputting light (e.g., emittingluminescence) over a greater number or range of wavelengths thanultimately desired as output light from the reference standard (i.e.,outputting light with both “desired” and “undesired” spectralcomponents). The subtractive element generally is configured to reduceor eliminate the undesired spectral components, thereby increasing therelative proportion of desired spectral components.

FIG. 5 shows an exemplary subtraction-based reference standard 500. Thisexemplary standard can include a support element 502, a relativelybroadband emissive element 504, a relatively narrowband subtractiveelement 506, an intensity filter element 508, a protective coating 510,and/or a mask (light-blocking element) 512, among others. These elementscan be discrete (e.g., separate, defined layers) and/or interdigitated(e.g., interwoven and/or blended layers (with shared or overlappingfunctions)). Typically, the subtractive and filter elements will bedisposed so that they will lie in use between the emissive element andthe detector, and the coating element will be disposed near an outersurface of the reference standard. More generally, these and otherelements of the subtraction-based (or other type of) reference standardcan be arranged in any suitable order.

Support element 502 generally comprises any material, or propertythereof, configured to provide structural support for the referencestandard. Exemplary support materials include glass and/or plastic,among others. The support element can take any suitable form, with anysuitable dimensions, such as a planar slab (e.g., sized like amicroscope slide, a gel, and/or a microplate, among others) and/or astandard sample holder (e.g., configured and sized like a microplate, abiochip, or the like).

Emissive element 504 generally comprises any mechanism for generating orproducing light over a greater number or range of wavelengths than thenumber or range(s) output by the reference standard (i.e., with desiredand undesired spectral components). The emissive element can producesuch light by any suitable mechanism, including transmission,reflection, absorption (generally selective absorption and/or absorptionand re-emission), and/or emission, among others. Transmission generallyincludes passing light through the element, unaltered, or altered viaabsorption, refraction, diffraction, scattering, and/or the like.Emission generally includes any mechanism for producing light within theemissive element, including photoluminescence and/or chemiluminescence,among others. Photoluminescent sources produce photoluminescence lightin response to illumination with suitable excitation light.Photoluminescence can include fluorescence (i.e., light produced by asinglet-to-singlet electronic transition) and/or phosphorescence (i.e.,light produced by a triplet-to-single electronic transition), amongothers. Chemiluminescent sources produce chemiluminescence lightassociated with a chemical reaction (e.g., as part of a reaction thatproduces an intermediary or product in an excited electronic state thatsubsequently decays by production of light) and/or electricalstimulation (e.g., electrochemiluminescence). Chemiluminescence caninclude bioluminescence (i.e., light produced by a biological reaction),among others. Suitable emissive elements can include luminophores,fluorophores, dyes, pigments, and/or the like. These elements can be wetor dry, liquid or solid, dissolved or suspended, homogeneous orheterogeneous, and so on.

Subtractive element 506 generally comprises any mechanism for alteringthe spectrum of the excitation and/or emission light, or portionsthereof. The spectral filter element generally acts to reduce oreliminate emission at selected wavelengths and/or over selected rangesof wavelengths (i.e., to reduce or remove undesired spectralcomponents). These functions can be accomplished via any suitablemechanism, using a single subtractive element or a combination ofsubtractive elements. Suitable subtractive elements include filterelements such as (1) short-pass (cut-off) filters, which passshort-wavelength light and reject long-wavelength light, (2) long-pass(cut-on) filters, which pass long-wavelength light and rejectshort-wavelength light, (3) bandpass filters, which pass light with aparticular wavelength (or range of wavelengths) and reject light withlower and higher wavelengths, and/or (4) band reject (or notch) filters,which reject light with a particular wavelength (or range ofwavelengths) and pass light with lower and higher wavelengths, amongothers. Short-pass and long-pass filters (also know as edge filters) canbe characterized by a cut-on or cut-off wavelength, among others, andbandpass and band reject filters can be characterized by a centerwavelength and a bandwidth, among others. Suitable subtractive elementscan include thin-film (e.g., metallic and/or interference) coatings,colored filter glass, holographic filters, liquid-crystal tunablefilters, and/or acousto-optical tunable filters, among others. Thesesubtractive elements can work by absorbing, reflecting, and/or bending(refracting or diffracting) light, among others. In some embodiments,the subtractive element can work by filtering portions of the excitationlight whose absorption gives rise to undesired spectral components ofthe emission.

Intensity filter element 508 generally comprises any mechanism foraltering the intensity of the excitation and/or emission light, orportions thereof. In some cases, the intensity filter element can reducethe overall intensity of the excitation light and/or emission light,substantially independent of wavelength. For example, the filter couldreduce overall excitation intensity to reduce photobleaching of thestandard, and it could reduce overall emission intensity to better matchcorresponding sample intensities, among others. In other cases, theintensity filter element can reduce the intensity of the excitationand/or emission light, according to wavelength. For example, the filtercould adjust the relative intensities of different spectral componentsof the emission light (e.g., making the height of multiple emissionpeaks more (or less) similar by using a filter element that blocksselectively a portion of the brighter (or dimmer) peaks). In any case,the strength of the intensity filter can be substantially uniform withposition, so that intensities from all portions of the referencestandard are reduced proportionally, or the strength of the intensityfilter can vary with position, so that intensities from some portions ofthe reference standard are reduced more than the intensities from others(e.g., to create a pattern). Suitable intensity filter elements includeneutral density filters, among others.

Protective coating 510 generally comprises any mechanism for protectingthe reference standard from unnecessary or unwanted exposure toenvironmental conditions. These conditions can include high humidity,harsh or corrosive chemicals, rough handling, and so on. Thus, theprotective coating can be relatively impervious to water and/or othercommonly encountered chemicals, and/or resistant to scratching, bending,and/or breaking, among others. Consistent with these functions, thecoating can be disposed about an outward or exterior surface of thereference standard, particularly on portions of the standard likely toencounter the unwanted condition(s). Suitable coating materials includeplastics, among others, particularly plastics that are at leastpartially transparent to excitation and/or emission light employedduring use of the reference standard.

Mask 512 generally comprises any mechanism for selectively rejectingmost or all of the emission from selected portions of the referencestandard. Suitable masks were discussed above; see particularly SectionI.C. The mask can be positioned to block excitation light and/oremission light, depending in part on the embodiment and intendedapplication.

Specific exemplary subtraction-based reference standards are describedbelow, in Example 1.

I.E Addition-Based Reference Standards

Addition-based reference standards generally comprise any referencestandards, or portions thereof, in which output light with the desiredspectral components is obtained by “adding” together or combining lightfrom different components that individually produce light having only asubset of the desired characteristics. Light from addition-basedreference standards can be used “as is” or spectrally filtered (as withthe subtraction-based reference standards) to remove any undesiredcomponents present after the addition. Such filtering can beaccomplished, among other ways, using a spectral filter that is integralto or otherwise associated with the standard and/or part of alight-detection system. Addition-based standards optionally can includeone or more of the various components described above forsubtraction-based standards, including a support, a filter element, acoating, and/or a mask, among others. Luminescent (and other) componentsof the addition-based reference standard can take any suitable form,including wet or dry, continuous or discrete, separate or blended,planar or formed, planar or sample-holder shaped, and/or so on. In someembodiments, the luminescent components can be selected and/or adaptedsuch that they can be excited using light with one wavelength (or rangeof wavelengths), while emitting at two or more distinguishablewavelengths (or ranges of wavelengths). Exemplary addition-basedreference standards can include photoluminescent nanocrystals (e.g.,“quantum dots”), luminescent glass, and so on. Specific exemplaryaddition-based reference standards are described below, in Example 2.

II. LIGHT DETECTION SYSTEMS

The light-detection system, as noted above, generally comprises anymechanism for detecting (and optionally analyzing) light from a sample.This system can include (1) a light source for producing light forilluminating, and/or inducing a suitable or desired response from, asample, (2) a sample-support device for supporting one or more samplesat an examination area, (3) a detector for detecting light transmittedor otherwise originating from a sample and optionally converting thedetected light into a representative signal, and/or (4) an optical relaystructure for directing and/or processing light between the light sourceand examination area, and/or between the examination area and thedetector, among others.

The components of the light-detection system can be selected and/orconfigured according to the intended application, desired level ofautomation, and so on. Typically, the light source produces light thatis directed by the optical relay structure along an incident opticalpath so that it impinges on or illuminates one or more samples disposedat one or more locations in the examination area. (This portion of thelight-detection system can be omitted or simply left unused inchemiluminescence applications.) Output light emitted, transmitted,reflected, scattered, and/or otherwise originating from the sample(s)then is directed by the optical relay structure along an output opticalpath onto the detector. The light-detection system can include anoptional controller adapted to control one or more system components,including, among others, the light source(s), the detector(s), and/or aregistration device configured to bring one or more samples and theexamination area into registration for analysis of the sample(s). Thecontroller can be configured to control or otherwise coordinate theillumination of samples and/or the detection of outputted radiation withthe relative position(s) of sample(s) and examination area. Thelight-detection system also can include additional components, such as(1) a fluidics mechanism to add, remove, and/or mix sample components,(2) a sample-handling mechanism to convey samples and/or sample holdersto and/or from the examination area and/or registration device, (3) ananalysis mechanism to analyze or interpret assay results, and/or (4) asample-identification mechanism such as a barcode reader to identifysamples and/or sample holders, and optionally to configure or operatesystem components accordingly, among others.

These and other aspects of light-detection systems provided by thepresent teachings are described below, including (A) light sources, (B)optical relay structures, (C) sample-support devices, (D) registrationdevices, (E) color separators, (F) detectors, (G) miscellaneous opticalelements, and (H) controllers, among others, as well as relationshipsand interactions there between.

II.A. Light Sources

The light source (102) generally comprises any mechanism for producinglight capable of illuminating, and/or inducing a suitable or desiredresponse from, a sample. For example, when used in an optical assay,light from the light source can, as a result of illuminating a sample,produce emitted (e.g., photoluminescence) light, transmitted light,reflected light, and/or scattered light, among others. These differentforms of light can be present exclusively, or in various combinations,and can include ultraviolet, visible, and/or infrared light, amongothers. The light source optionally can induce a similar or relatedresponse from a suitably positioned optical reference standard.

Exemplary light sources can include continuous wave and pulsed lasers,arc (e.g., xenon) lamps, incandescent (e.g., tungsten halogen) lamps,fluorescent lamps, electroluminescent devices, laser diodes, and/orlight-emitting diodes (LEDs), among others. Such light sources can becapable of use in one or more illumination modes, including continuousand/or time-varying (e.g., pulsed or sinusoidally varying) modes, amongothers, depending on system configuration and/or intended application.For example, an arc lamp or continuous wave laser can be used to providecontinuous illumination, and a pulsed laser can be used to provideintermittent illumination. Such light sources also can produce coherent,incoherent, monochromatic, polychromatic, polarized, and/or unpolarizedlight, among others. For example, an arc lamp can be used to provide (atleast initially) incoherent, polychromatic, unpolarized light, and alaser can be used to provide (at least initially) coherent,monochromatic, polarized light, among other possibilities.

The light source can be used alone but would be used more commonly incombination with various optics and/or other mechanisms (such as theoptical relay structures described below). These optics and/or othermechanisms can be used to alter the nature of the light output by thelight source (e.g., its color (spectrum or chromaticity), intensity,polarization, and/or coherence, among others). Alternatively, or inaddition, these optics and/or other mechanisms can be used to directand/or alter the size, shape, and/or numerosity of the light beam(s)(e.g., to illuminate selected locations in an examination area with oneor more light beams). The light that ultimately is incident on thesample(s) can be produced by one or more light sources, and can bedirected and/or modified by one or more optical devices operativelydisposed between the light source(s) and the examination area. Theresultant light beam or beams can be one or more of various forms,including but not limited to diverging, collimated, and converging,among others. This beam or beams can be directed onto an examinationarea in a manner inducing light production from one or more sampleslocated in a plurality of locations within the examination area.

II.B. Optical Relay Structures

The optical relay structure (108) generally comprises any mechanism(s)for directing, transmitting, and/or conducting light between two points,such as from a light source toward a sample (or examination site) and/orfrom a sample (or examination site) toward a detector. These structurescan stand alone and/or be portions of or integral to other systemcomponents, such as the light source, color separator, and/or detector,among others. The optical relay structures can be configured to directone or more light beams, in the same or different directions, along thesame, multiple, or different optical paths.

The optical relay structures can include any suitable combination ofoptical elements. These elements independently can be part of a single(e.g., excitation or emission) relay structure, or can be shared betweentwo or more relay structures. Exemplary optical elements can include (1)reflective elements, such as concave, planar, and/or convex mirrors,among others, (2) refractive elements, such as converging, diverging,concave, convex, and/or planoconvex lenses, including circular and/orcylindrical lenses, among others, and/or (3) transmissive or conductiveelements, such as glass or quartz fiber optics and/or liquid lightguides, among others.

The optical relay structure(s) can be selected, in conjunction with thelight source(s) and/or detector(s), to allow any suitable or desiredcombinations of illumination and/or detection. For example, thesecomponents can be arranged to allow same-side, (locally) anti-parallelor straight-on (“epi”) illumination and detection, such as topillumination and top detection, or bottom illumination and bottomdetection, respectively. Alternatively, or in addition, these componentscan be arranged to allow opposite side, (locally) parallel orstraight-through (“trans”) illumination and detection, such as topillumination and bottom detection, or bottom illumination and topdetection, respectively. Alternatively, or in addition, these componentscan be arranged to allow illumination and/or detection at obliqueangles. For example, illumination light can impinge on the bottom of asample holder at an acute angle (e.g., about 45 degrees) relative todetection. Such oblique illumination and detection can reduce the amountof excitation light reaching the detector, relative to straight-on episystems (light source and detector directed at about 90 degrees tosample holder) or straight-through trans systems (light source directedthrough a sample holder directly at a detector). Epi systems areespecially suitable for photoluminescence assays, trans systems areespecially suitable for absorbance assays, and oblique systems (with theincidence angle set above the critical angel) are especially suitablefor total internal reflection assays, among others.

II.C. Sample-Support Devices

The sample-support device (104) generally comprises any mechanism forsupporting one or more samples at a sample site in an examination area.In typical embodiments, the sample-support device allows for the receiptand/or transmission of light relative to one or more samples supportedby the device. General examples of suitable sample-support devices caninclude trays, wells, tubes, containers, channels, chambers, frames,carriages, holders, slides, shelves, stages, housings, and/or the like.Specific examples of suitable sample-support devices can includemicroplates, PCR plates, microtiter plates, cell culture plates,biochips, hybridization chambers, chromatography plates, and/ormicroscope slides, among others. Specific locations in thesample-support device, such as wells in microplates, PCR plates,microtiter plates, and cell culture plates and array sites on biochips,can comprise assay sites. For example, microplates (and/or PCR plates,microtiter plates, and/or cell culture plates) can include arrays of 6,12, 24, 48, 96, 384, 864, 1536, 3456, and/or 9600 such assay sites,among others. The sample-support devices can be configured to allow topdetection (e.g., by having an open or at least partially transparenttop), bottom detection (e.g., by having an at least partiallytransparent bottom), and/or side detection (e.g., by having an at leastpartially transparent side), among others. In some embodiments, thesample-support device can be configured, additionally and/oralternatively, to support an optical reference standard, for use before,during, and/or after sample analysis.

II.D. Registration Devices

The registration device generally comprises any mechanism for bringing asample(s) and an examination area into registration, for analysis of thesample(s).

The registration device can move the sample (or associatedsample-support device), the examination area, and/or both. For example,to effect relative movement of a sample and examination area, theregistration device can include a driver, such as a stepper motor, thatmoves a carriage, tray, conveyor, stage, frame, and/or other structureor structures adapted to support the sample, an associatedsample-support device, and/or a detector, among others. The registrationdevice can move the sample and/or examination area in any suitable formor combination of motion(s), including, among others, (1) continuous orintermittent, (2) unidirectional, bidirectional, or multi-directional,and/or (3) rectilinear or curvilinear. Such movement can occur in the x,y, and/or z directions, and can occur parallel, perpendicular, and/orskewed to the excitation and/or emission axes.

The registration device can be under manual and/or automated control.Automated control can be particularly useful for analysis of pluralsamples, allowing successive registration of multiple samples and theexamination site. Such control can be effected using any suitablemechanism, for example, moving a sample-support device and/orexamination area in preselected increments and/or until preselectedcriteria are fulfilled. Such preselected increments can correspond topredefined separations between samples, or sample sites, as found in amicroplate, PCR plate, biochip array, and/or the like. Such preselectedcriteria also can correspond to indicia (such as increased intensity)indicative of the presence of a sample, as found in bands on aseparatory gel or column, among others. Such preselected criteria alsocan correspond to the positions of reference markings on an opticalreference standard (e.g., portions 314 in FIG. 3 and/or portions 406 and410 in FIG. 4, among others).

II.E. Color Separators

The color separator generally comprises any mechanism for spatiallyseparating, or distributing, light according to its wavelengthcomposition (or spectrum).

The color separator can use any suitable mechanism(s) and/orcomponent(s) for separating light. Exemplary mechanisms can includediffraction, interference, and/or refraction, among others. Exemplarycomponents can include (diffraction) gratings, interferometers, and/orprisms, among others. In some embodiments, the color separator caninclude two or more sequentially acting components, employing the sameor different mechanisms, with the first component achieving a coarsecolor separation, and the second component achieving a finer or finalcolor separation.

The color separator can separate multi-wavelength light by directinglight with different wavelengths along different paths (e.g., indifferent directions, at different angles, etc.) The separation can bepartial or complete, and can create bands or beamlets of light, whichcan be continuous or discrete, and which can be partially overlapping orcompletely distinct. The character of the separation typically will bedetermined at least in part by the character of the light beingseparated. Thus, input light with several well-spaced wavelengthcomponents can give rise to separated output light at several discrete(well-spaced) positions, while input light with closely or continuouslyspaced wavelength components can give rise to separated output lightover a continuous set of positions.

The separated light generally can form any distinguishable pattern.Thus, the separated light can form a linear array, in which thewavelength of light varies with position along the line. Alternatively,the separated light can form a circular array, in which the wavelengthof the light varies with distance from the center of the circle. In someembodiments, the color separator can produce light that is spectrallyseparated along a first axis, and spatially differentiated along asecond axis at least substantially transverse to the first axis. Inthese embodiments, position along the first axis provides informationabout the spectral composition of light emitted from the sample, andposition along the second axis provides information about the positionfrom which the spectrally separated light arose in the sample.

The separated light can be directed onto a common detector, ontoseparate detectors, or onto a combination of detectors. The relationshipbetween wavelength and position on the detector(s) can be determinedempirically, for example, using input or calibration light of knownwavelength(s). Alternatively, or in addition, the relationship betweenwavelength and position on the detector(s) can be determinedtheoretically, for example, by calculating the optical paths for lightof different wavelengths. The position(s) of light on the detector canbe determined by a variety of factors, including (1) the mechanism usedto separate the light, (2) the angle(s) at which the light leaves thecolor separator, the distance between the color separator and thedetector(s) (generally, greater distances between the color separatorand detector(s) will give rise to greater separations between light ofdifferent wavelengths on the detector(s)), and/or (3) the type ofpattern (e.g., linear versus circular) formed by the separated light,among others.

The spatial distribution or pattern of detected light can be convertedinto information about the distribution and identity(ies) of componentsof the sample, using any suitable method. These methods can includesimply looking up a result in a look-up table (e.g., position (x,y) onthe detector corresponds to light of wavelength λ (or wavelengths withinsome extended range (e.g., λ₁ to λ₂)) emitted from position (X,Y) (orpositions within some extended range) in the sample, evaluating afunction expressing the relationship between these parameters, and/orthe like. The desired result can be obtainable simply by notingqualitatively the presence or absence of light at a particular positionon the detector (subject, in some cases, to some threshold amount), orit can be obtainable by determining quantitatively the amount of light(intensity, number of photons, amount of energy, etc.) detected at theposition, among others.

The color separator can be disposed, in some embodiments, so that itacts only on light directed from a sample toward a detector, withoutacting on (or, in most cases, even contacting) light directed from alight source toward a sample.

The color separator can be included, in some embodiments, as part of aspectroscope or other instrument for producing and observing spectrafrom light or other electromagnetic radiation emitted from a sample.

Exemplary color separators are described further in U.S. ProvisionalPatent Application Ser. No. 60/584,525, filed Jun. 30, 2004, titledDistributed Light Detector of inventors Charles S. Vann and StevenBoege, which is incorporated herein by reference.

II.F. Detectors

The detector (106) generally comprises any mechanism for detecting lighttransmitted or otherwise originating from a sample and optionallyconverting the detected light into a representative signal.

Exemplary detectors can include film, charge-coupled devices (CCDs),intensified charge-coupled devices (ICCDs), charge injection device(CID) arrays, videcon tubes, photomultiplier tubes (PMTs),photomultiplier tube (PMT) arrays, position sensitive photomultipliertubes, photodiodes, and/or avalanche photodiodes, among others. Suchdetectors can be capable of use in one or more detection modes,including (1) imaging and point-reading modes, (2) discrete (e.g.,photon-counting) and analog (e.g., current-integration) modes, and/or(3) steady-state and time-resolved modes, among others. Particularly ifused with a color separator, the detectors can be configured to receivea two-dimensional array of light, which may be separated along a firstdimension according to position in a sample or sample array, and along asecond dimension according to spectral composition. Toward this end, thedetector can include bins for detecting light of different colors, forexample, corresponding to light from different luminophores. These binscan be the same or different sizes, and can be formed of one or moresub-bins, or pixels, depending in part on the average separation betweenspectral peaks output by the color separator.

The detector can be used alone or in combination with various opticsand/or other mechanisms (such as the optical relay structure describedabove). These optics and/or other mechanisms can be used to alterproperties of the light (e.g., color, intensity, polarization,coherence, and/or size, shape, and/or numerosity of the light beam(s),as described elsewhere herein), prior to its detection. In someembodiments, the detector can be part of or coupled to a spectrograph orspectroscope for analyzing the spectral composition of the detectedlight.

II.G. Miscellaneous Optical Elements

The light-detection system, and/or components thereof, also can includemiscellaneous optical elements capable of performing additional and/orduplicative optical functions. These optical elements can include (1)intensity filters (such as neutral density filters) for reducing theintensity of light, (2) spectral filters (such as interference filters,diffraction gratings, and/or prisms) for altering or selecting thewavelength(s) of light (e.g., for separating longer-wavelength emissionlight from shorter-wavelength excitation light, in single-photonphotoluminescence, and/or for separating shorter-wavelength emissionlight from longer-wavelength excitation light, in multi-photonphotoluminescence), (3) polarization filters (such as “polarizers”) foraltering or selecting the polarization of light, (4) “confocal opticselements” (such as an aperture or slit positioned in an intermediateimage plane) for reducing or eliminating out-of-focus light, (5) beamcollimators for converting input light (particularly diverging inputlight) into an at least substantially collimated light beam, (6) beamexpanders for increasing the cross-sectional area of a beam of light,(7) beam homogenizers (such as a fiber optic cable or liquid lightguide) for enhancing the spatial uniformity of light, and/or (8)reference monitors for correcting for variations (e.g., fluctuationsand/or inhomogeneities) in light produced by a light source and/or otheroptical elements. These elements can be functional in one or more of thespace, time, and/or frequency domains, as necessary or desired.

The relative positions of any intensity, spectral, polarization, and/orother optical elements generally can be varied without affecting theoperation of the light-detection system. In addition, if there is morethan one optical path, for example, to permit top and bottom or obliqueillumination and/or detection, optical elements can be shared and/orused independently in each path. The particular order, positions, andcombinations of optical elements for a particular experiment can dependon the apparatus, the assay mode, and the sample (target material),among other factors. In some cases, optical elements can be associatedwith an exchange mechanism, such as a wheel or slider, that allowsconvenient and automatable placement and exchange of optical elements byrotating, sliding, or otherwise bringing preselected optical elementsinto or out of the optical path.

II.H. Controllers

The controller generally comprises any mechanism for controllingcomponents and/or other aspects (including calibration and/ormonitoring) of the light-detection system. These components and/or otheraspects can include the light source, optical relay structures,registration device, detector, and/or optical reference standard, amongothers. For example, the controller can determine and/or change (1) thewavelength, intensity, and/or (spatial and/or temporal) uniformity oflight produced by the light source, (2) the order and timing of sampledelivery by the registration device and image acquisition by thedetector, and/or (3) the wavelength and/or intensity of light detectedby the detector, among others. The controller can include hardware,software, firmware, and/or a combination thereof, and can be any device,or combination of devices, adapted to store and execute instructions tocontrol associated detection system components. The controller caninclude one or more of various devices, such as a computer, computerserver, microprocessor, memory, logic unit, and/or processor-basedsystem capable of performing a sequence of logic operations. Inaddition, processing can be centralized (with two or more componentssharing a common controller) and/or distributed (with one or morecomponents having their own dedicated controllers, acting alone, orconnected to one another and/or a central controller).

III. APPLICATIONS

Light-detection systems calibrated and/or monitored using opticalreference standards in accordance with the present teachings can be usedfor any suitable purposes, such as detecting and/or monitoring theoccurrence of, and/or changes in, light or other forms of radiationreceived from one or more suitable samples.

The detection or monitoring of light can be performed qualitativelyand/or quantitatively. Qualitative detection can include measurement ofthe presence or absence of a signal, and/or a change in a signal frompresent to absent, or absent to present, among others. Here, presence orabsence can be in reference to a whole signal (such as any light) and/ora component of the signal (such as light of a particular wavelength,polarization, and/or the like). The signal, and/or components thereof,can arise from the sample itself and/or from labels (or other reporters)attached to or otherwise associated with the sample. Quantitativedetection can include measurement of the magnitude of a signal, such asan intensity, wavelength, polarization, and/or lifetime, among others.The quantified signal can be used alone and/or compared or combined withother quantified signals and/or calibration standards. The standard cantake the form of a calibration curve, a calculation of an expectedresponse, and/or a control sample measured before, during, and/or aftermeasurement of the sample of interest.

The detected or monitored light can be used for any suitable purpose,for example, to determine the presence, absence, amount, concentration,activity, and/or physical properties (including interactions) of ananalyte (such as a photoactive analyte) in a sample. Here, the analytecan be the actual moiety of interest and/or a reporter moiety (such as aluminophore) that reports on the actual moiety of interest.

The moiety of interest can be a reaction component. Exemplary reactioncomponents can include an enzyme, enzyme substrate, enzyme product,and/or enzyme modulator (e.g., agonist and/or antagonist). Suitablereactions can occur in vivo and/or in vitro, for example, as part of acell-lysis experiment and/or a polymerase chain reaction (PCR)preparation. Exemplary reaction components also can include precursorsand/or products of a synthetic pathway, such as an amino acid, peptide,protein, nucleotide, oligonucleotide, nucleic acid polymer,carbohydrate, fatty acid, lipid, and/or the like.

The moiety of interest also can be the subject and/or product of aseparatory process, such as on a chromatograph, gel, column, and/or thelike. Here, the separatory process can include single processes, such ascolumns giving rise to fractions, and/or multiple processes, such asparallel lanes on a gel giving rise to sets of bands.

The moiety of interest also can be the subject of a sequencing process,such as a peptide, protein, oligonucleotide, and/or nucleic acid (RNA orDNA) sequencing process. Here, the sequence can include amino acidsequence, nucleotide or base sequence (e.g., G, C, T, A, U, etc.), andso on, and the sequencing process can include generating fragments (orother derivatives) of the moiety to be sequenced and labeling thosefragments (before or after their generation) with differentluminophores. Thus, in nucleic acid sequencing, the presence of a G, C,T, A, or U at a particular position in a moiety of interest, or in afragment or derivative thereof, can be determined by the identity of anassociated luminophore.

The moiety of interest also can be the subject of an identification, oraffinity, process, such as a northern, western, and/or southern blot.

In some cases, the effect of some condition on the moiety of interestcan be determined, for example, by comparing results in the presence ofthe condition with predicted and/or measured results in the absence ofthe condition and/or the presence of another condition. Exemplaryconditions can include presence or absence of a modulator (agonist orantagonist) or cofactor, and/or changes in temperature, concentration,pH, osmolarity, ionic strength, and/or the like.

The sample can include any appropriate material, with any suitableorigin. For example, the sample can include and/or be derived from abiomolecule, organelle, virus, cell, tissue, organ, and/or organism. Thesample can be biological in origin and/or synthetically prepared. Asample optionally can be, or can be derived from, a biological sample,such as a sample prepared from a blood sample, urine sample, fecalsample, saliva sample, and/or mucous sample, obtained using any suitablephysiological sampling method, such as a swipe or a smear, among others.A sample optionally can be, or can be derived from, an environmentalsample, such as an air sample, a water sample, or a soil sample. Asample can be aqueous, and yet can contain biologically compatibleorganic solvents, buffering agents, inorganic salts, or other componentsknown in the art for assay solutions. Suitable samples (or compositions)can include compounds, mixtures, surfaces, solutions, emulsions,suspensions, cell cultures, fermentation cultures, cells, suspendedcells, adherent cells, tissues, secretions, and/or derivatives and/orextracts thereof. Depending on the assay, the term “sample” can refer tothe contents of a single sample site (e.g., microplate well) or of twoor more sample sites.

IV. EXAMPLES

The following examples describe selected aspects of the presentteachings. These selected aspects include, among others, exemplaryapparatus, methods, and compositions for detecting light. The selectedaspects can be combined with other aspects in the same and/or otherexamples, and/or in other portions of these teachings, as suitableand/or desired. These examples are included for illustration and are notintended to limit or define the entire scope of the disclosed concepts.

Example 1 Exemplary Subtraction-Based Reference Standards

This example describes an exemplary subtraction-based reference standard500, in accordance with aspects of the present teachings; see FIGS. 5-9.The exemplary standard can be used with multi-luminophorephotoluminescence systems, among others, including but not limited tocarboxyfluorescein (FAM) and carboxy-X-rhodamine (ROX) (or similar greenand red emitting) dual-luminophore systems.

FIG. 5 is a schematic sectional view of reference standard 500. Thisexemplary standard, which was described above in more detail in SectionI.D, can include several components: (1) a support element 502, (2) abroadband photoluminescent element 504, (3) at least one subtractive orspectral filter element 506 a,b,c, (4) an intensity filter element 508,(5) a coating element 510, and/or (6) a light-blocking element (or mask)512, among others. Here, these components are arranged as a series of atleast substantially planar layers, in the indicated order; however, moregenerally, these components can have any suitable form, and any suitableorder. Moreover, here, the broadband photoluminescent component includesUltem® 1000 polyetherimide (PEI) plastic polymer, and the spectralfilter components include cold-coating filter materials configured totransmit light with FAM and ROX spectra. Overall, the reference standardcan take any suitable form, for example, a 3.10-inch width×6.60-inchlength× 1/16-inch height sheet, corresponding to the approximate size ofa Society for Biomolecular Screening (SBS) standard microplate used incommon fluorescence detection systems, among others.

FIG. 6 is a plot of an exemplary emission spectrum for broadbandphotoluminescent component 504, from reference standard 500. Theemission spectrum is a plot of the relative intensity ofphotoluminescence emitted from the photoluminescent component as afunction of the wavelength of the photoluminescence, followingexcitation with suitable excitation light (i.e., excitation light havinga wavelength (or range of wavelengths) capable of inducingphotoluminescence emission from the photoluminescent component). Thespectrum represents photoluminescence emitted by uncoated Ultem® 1000plastic, following excitation at 488 nm (e.g., using an argon ionlaser). Ultem® 1000 plastic can be translucent, rather than opticallyclear, with a yellow tint and a broad photoluminescence emissionspectrum. In addition, Ultem® 1000 plastic can have one or moreadvantageous mechanical properties, such as durability and/orhigh-melting temperature, among others. The spectrum shows an envelopeof values constructed by displaying the highest and lowest values ateach wavelength, based on measurements of 384 similarly preparedsamples.

FIG. 7 is a plot of individual and composite transmission spectra forfilter elements 506 a,b,c, from reference standard 500. The transmissionspectra are plots of relative transmissivity as a function ofwavelength. In these spectra, higher values of the transmissivitycorrespond to relatively greater transmission (and lesser absorption) oflight, and lower values of the transmissivity correspond to relativelylesser transmission (and greater absorption) of light. Here, there arethree filter layers, each with its own transmission spectrum: (1) layer1 (dashed line), a short-pass filter, that generally transmitsshort-wavelength light, and generally blocks long-wavelength light, (2)layer 2 (thin solid line), a first band-reject (or notch) filter, thatgenerally rejects light at a first intermediate wavelength, whilegenerally passing light with relatively shorter and longer wavelengths,and (3) layer 3 (dot-dashed line), a second band-reject (or notch)filter, that generally rejects light at a second intermediatewavelength, while generally passing light with relatively shorter andlonger wavelengths. The combined or composite action of these threefilter layers (thick solid line) creates a bandpass filter thatgenerally transmits light at two distinct wavelengths (or ranges ofwavelengths) and that generally blocks light with other (nearby)wavelengths. For example, in a FAM/ROX dual-luminophore system, theoptical coating typically will have a relatively narrow bandpasstransmission peak near the emission band of each luminophore, that is,near about 520-nm (green) wavelength for the FAM dyes, and near about600-nm (red) wavelength for the ROX dye, among others. Moreover, ifexcitation and emission occur on the same side of the referencestandard, as in epi-fluorescence mode, the combined or compositionaction of the three filter layers also can pass light at wavelengthsshorter or longer than those of the two transmission peaks, forsingle-photon or multi-photon excitation, respectively. For example, forsingle-photon excitation of FAM and ROX dyes, the optical coatingtypically will pass light near about 488-nm (blue-green) wavelength.Suitable filter layers are available commercially. For example, suitablecold coatings can be obtained from Chroma Technology Corp. (Rockingham,Vt.) for application to the support and/or broadband component.

FIG. 8 is a plot of an exemplary calculated emission spectrum forreference standard 500. The calculated emission spectrum (solid line) isa product of the emission spectrum of broadband photoluminescentcomponent 504 (thick dashed line; see also FIG. 6) and the compositetransmissivity of filter components 506 a-c (thin dashed line; see alsoFIG. 7).

FIG. 9 is a plot of an exemplary measured emission spectrum forreference standard 500. The spectrum, measured on an Applied Biosystems(AB) Sequence Detection System 7900 instrument, includes two distinct,relatively narrow peaks, as predicted in FIG. 8. In this embodiment, theintensities of these peaks are about one-sixth those of the broadbandcomponent alone; however, in other embodiments, the intensities can beincreased using other substrate materials and/or filter components. Thisspectrum, like that in FIG. 7, shows an envelope of values defined bythe highest and lowest values at each wavelength, based on measurementsof 384 wells.

Example 2 Exemplary Addition-Based Reference Standards

This example describes exemplary addition-based reference standards, inaccordance with aspects of the present teachings; see FIGS. 10 and 11.The exemplary standards can be used with multi-luminophorephotoluminescence systems, among others, including but not limited tocarboxyfluorescein (FAM) and carboxy-X-rhodamine (ROX) (or similar greenand red emitting) dual-luminophore systems. These standards generallycan include any combination of two or more luminophores, with differentemission spectra, and optionally with similar or overlapping excitationspectra (such that the combinations of luminophores can be excited usinglight of similar wavelength). The exemplary standards can include (A)photoluminescent nanocrystals (“quantum dots”) and/or (B)photoluminescent glass, among others, as described below.

A. Photoluminescent Nanocrystal-Based Reference Standards

This section describes exemplary photoluminescent nanocrystal-basedreference standards; see FIG. 10. Photoluminescent nanocrystals, as usedherein, generally can include any small, solid-state photoluminescentcompositions, including but not limited to “quantum dots.”

Photoluminescent nanocrystals can include (1) a core, (2) a shell, and(3) an optional coating, among others. The core typically is the primarysource of the nanocrystal's photoluminescence, with the composition andsize of the core (primarily) determining the properties of thisphotoluminescence. The composition of the core can coarsely determineemission properties. For example, cadmium sulfide (CdS) cores can beparticularly suitable for ultraviolet-blue emission, cadmium selenide(CdSe) can be particularly suitable for visible emission, and cadmiumtelluride (CdTe) can be particularly suitable for far-red andnear-infrared emission, among others. The size of the cores can morefinely determine emission properties. For example, relatively large(>6-nm diameter) CdSe cores can be used to prepare 655-nm emittingnanocrystals, and relatively small (<3-nm diameter) CdSe cores can beused to prepare 525-nm emitting nanocrystals. The cores generally canhave any suitable shape, including spheres, rods, and/or pyramids, amongothers. The shell typically surrounds the core, functioning tostrengthen and stabilize photoluminescence emission. The shell can beformed of any suitable material(s). The coating typically surrounds theshell (and thus the core), functioning to determine the hydrophilicityand/or reactivity of the nanocrystal, among others. The coating can beformed of any suitable material(s), including hydrophobic and/orhydrophilic materials, reactive groups, binding groups (e.g., antibodiesor antigens. avidin or biotin, lectins or sugars, etc.), and so on.

Photoluminescent nanocrystal-based optical reference standards (likeother optical reference standards in accordance with the presentteachings) can take any suitable format, including, among others, (1)discrete formats, in which the luminophores are disposed at discretesites (e.g., corresponding to microplate wells), and/or (2) continuousformats, in which the luminophores are disposed continuously (e.g., inand/or on a microscope slide), as described below.

A.1. Discrete Embodiments

Discrete formats correspond to reference standards in which theluminophores are disposed at discrete sites in and/or on the referencestandard. Exemplary discrete formats can include microplate-basedembodiments, among others, including wet and dry embodiments. In wetembodiments, soluble or suspendable luminophores such asphotoluminescent nanocrystals can be dissolved or suspended in anappropriate (e.g., aqueous) solution, before or after being dispensedinto wells of a microplate. Suitable microplates can have 96, 384,and/or 1536 wells, among others. Wet microplate-based standards couldprovide various benefits. For example, wet standards could position theluminophores in the reference standard in the same or similarenvironment (solution and/or sample holder) as the luminophore(s) in thesample, to account for any contribution to the signal from the sampleholder. Moreover, wet standards could position the luminophores in thestandard at the designed focal point of the optical detection systemused in the assay.

Discrete (microplate and/or other well or fluid based) embodiments canbe covered or sealed, using any suitable mechanism and material, toincrease their robustness and durability. Suitable seal mechanisms caninclude pressure-sensitive adhesive (PSA), heat seal material (where oneor more layers melts, flows, and bonds to the microplate (or othersample holder)), ultrasonic welding, and/or similar devices andprocesses. Exemplary embodiments can include a silicone-based adhesiveon a clear polyolefin laminate backing (polypropylene/polyethyleneterephthalate (PP/PET)) with a white polyester film release liner,and/or a heat-sealable polyester-based film laminate of PET/MR, in whichthe seal optionally can fuse with the microplate material. In someembodiments, with or without a cover or seal, an evaporation inhibitorsuch as DMSO can be added to the aqueous solution to further preventevaporation. In these embodiments, if any air leaks do occur in theseal, the DMSO will act to absorb ambient moisture.

Luminophores can be suspended, in some embodiments, in a low-fluorescinggel, polymer, epoxy, and/or other solid or semi-solid material medium,before or after being dispensed into a well or other embodiment. Thefluid compartment can again be sealed, transiently or permanently, by anacceptable sealing material. The suspension material and optionalsealing would lower the risk of unwanted leaks and spills. Specificexamples of alternative suspension media include polymer gels, agarosegels, acrylamide gels, optical grade epoxies, and/or others.

Luminophores can be “dried down,” in some embodiments, after beingdispensed—suspended or dissolved—into a fluid compartment, byevaporating off the carrier solvent(s). The dried-down luminophores,such as photoluminescent nanocrystals, still would exhibit their stablefluorescence spectra, but without the risk of liquid spills. Thisapproach is particularly suitable as a standard for adherent cellassays, because it positions the luminophores in the standard at thesame focal height as the cells in the assay. The microplates could besealed, transiently or permanently, by an acceptable seal material, asabove.

FIG. 10 is an exemplary emission spectrum for an addition-based,photoluminescent-nanocrystal-containing reference standard, showingrelative emission intensity as a function of emission wavelength,following excitation at a fixed excitation wavelength. The shadedenvelope is constructed from the lowest and highest intensities measuredat each emission wavelength, based on analysis of 20 identicallyprepared samples. Here, exemplary photoluminescent nanocrystals withemission at 525 nm and 608 nm were mixed and suspended in aqueoussolution, and pipetted into wells in a 384-well microplate, formeasurement of the emission spectrum. Specifically, the 525-nm and608-nm nanocrystals were suspended at concentrations of 0.55 μM and0.034 μM, respectively, and then 10 μL of the resulting solution wasadded to each well measured. The molar concentration of the suspendednanocrystals optionally can be correlated or matched to the appropriatemolar concentrations of dyes, such as FAM and ROX dyes, commonly used inbiological assays. Suitable microplates (for any discrete embodiments)can include, among others, the AB 96-well microplate, the AB 384-wellmicroplate, and the AB 384-well Micro Fluidic Card (MFC) (AppliedBiosystems, Foster City, Calif.). Suitable light-detection systems (forany embodiments) can include, among others, the AB Sequence DetectionSystems Models 7000, 7300, 7500, 7700, and 7900 (Applied Biosystems),among others.

In various embodiments, instruments for thermal cycling can include aheated lid with apertures corresponding to wells to be interrogated anda thermal block to provide thermal contact with the wells. Theluminescence reference plate can contact to the block to establish theappropriate temperature for the light-producing elements. Theluminescence reference plate includes an insulator on the upper side ofthe carrier with light-blocking elements to prevent contact with theheated lid that can avoid thermal short. The carrier can be configuredas apertures on the top portion forming the light-blocking elements. Theapertures in the carrier can be configured to align with the aperturesin the heated lid. The carrier can be configured with recesses on thebottom portion where the recesses on the bottom portion correspond tothe apertures on the top portion. The top portion and bottom portion ofthe carrier can be configured to bound a layer of media forming thelight-producing elements. The recesses on the bottom portion of thecarrier can include black paint to provide a background correction. Auser of the luminescence reference plate can obtain the luminescentcalibration from the top portion on the carrier and then flip theluminescence reference plate over to obtain a black backgroundcorrection.

A.2. Continuous Embodiments

Continuous formats correspond to reference standards in whichluminophores are disposed continuously throughout part or all of thereference standard. This “continuous” distribution can be uniform orvariable, in one, two, or three dimensions.

Exemplary embodiments can include (1) a support element, such as aplanar support element, and (2) a light-producing layer (or layers) offluorescence reference standard material, such as photoluminescentnanocrystals. The light-producing layer(s) can be formed on an outersurface of the support element, and/or sandwiched between layers (orother portions) of the support element. The support element and/orlight-producing layer(s) optionally can be covered with a coating, forexample, as described above.

The light-producing layer(s) can be produced by any suitable mechanism,such as a solid layer than can be transferred intact to the supportstructure (like a cellophane), or as a liquid or vapor applied to orcontacted to the support element. For example, in the latter case, toproduce a thin sheet of fluorescence reference standard material with acontinuous and uniform fluorescent surface, the luminophores can besuspended in an appropriate solution and spin coated onto the supportelement. For a glass substrate, appropriately charged luminophores canbe spin coated and adhered onto the glass surface. For a more durablesolution having a plastic substrate, luminophores that are soluble, orsuspended, in organic solvents can be used. The appropriately coatedluminophores can be suspended in organic solvents and put into afluorinated polymer. In this case, the organic solvents can be driedoff, leaving the luminophores in the plastic polymer.

B. Photoluminescent-Glass-Based Reference Standards

This example describes exemplary photoluminescent glass referencestandards; see FIG. 11. Glasses, as used herein, generally can includeany super-cooled liquids or noncrystalline solids, with intrinsicluminescence and/or with “added” luminophores. The glass can be used asa continuous slab, alone or in conjunction with a mask that limitsexcitation and/or emission to discrete locations. Alternatively, or inaddition, the glass can be disposed at discrete locations, for example,in microplate wells. Glasses can have a number of potential advantages,including sensitivity (efficiency), durability (e.g., to temperaturesabove 100° C.), wide excitation band, large Stokes' shift, and so on.The number of bands and the relative intensities of the bands can beadjusted by using suitable filter materials (just like with thesubtraction-based standards).

FIG. 11 shows an exemplary emission spectrum obtained with Lumilass G9™glass, a commercially available photoluminescent glass (SUMITA OpticalGlass, Inc.). Suitable photoluminescent glasses can include (1) LumilassR7™ glass, which can be excited with ultraviolet light in the 200-420 nmrange, and which emits red fluorescence at about 610 nm, among others,(2) Lumilass G9 glass, which can be excited with ultraviolet light inthe range 200-390 nm, and which emits green florescence at about 540 nm,among others, and (3) Lumilass B™ glass, which can be excited withultraviolet light in the range 200-400 nm, and which emits bluefluorescence at about 405-410 nm, among others.

Example 3 Exemplary Light-Detection System

This example describes an exemplary light-detection system 1200,including an associated optical reference standard, in accordance withaspects of the present teachings; see FIG. 12. This exemplary embodimentis described with reference to specific components; however, thesecomponents can be omitted or substituted with other components, asappropriate or feasible.

Light-detection system 1200 includes (1) a light source 1202 (e.g., avertically oriented tungsten halogen lamp), (2) a detector 1204 (e.g., acharge-coupled device (CCD) camera), and (3) an optical relay structure1206 for directing light from the light source onto one or more samples1208 disposed in wells 1210 of a multiwell sample holder 1212 such as amicroplate at an examination site 1214, and from the sample(s) to thedetector. The optical relay structure, in the pictured embodiment,includes a (long-pass) dichroic beamsplitter 1216 for separating andproperly routing excitation and emission light, and a fold mirror 1218for turning excitation and emission light toward and away from thesamples, respectively. The light-detection system further may include aninfrared-blocking “hot mirror” 1220 for filtering out infrared light, anexcitation filter 1222 for filtering out additional unwanted (e.g.,selected visible) excitation light, a Fresnel lens 1224 and well lenses1226 for directing light onto the samples, and an emission filter 1228for filtering out stray excitation light mixed with the emission light.

The light-detection system optionally includes and/or can be used withan optical reference standard 1230. This reference standard ispositioned, in the pictured embodiment, in the optical path between thefold mirror (1218) and the Fresnel lens (1224). The reference standard,which resembles reference standard 300 (embodiment “a”) in FIG. 3,includes a light-producing component 1232 and a light-blocking or maskcomponent 1234. The reference standard functions like a multi-wellversion of optical reference standard 110 in FIG. 1.

Example 4 Miscellaneous Matters

This example describes miscellaneous aspects of light-detection systemsand optical reference standards, in accordance with aspects of thepresent teachings.

The optical reference standard can be used for any suitable purpose,consistent with these and/or other applications. These purposesoptionally can include, among others, (1) focusing images of microplatewells onto a CCD camera by adjusting a focusing lens for maximumfluorescence intensity from the reference standard, (2) rotating a CCDcamera so that pixel columns are parallel to the rows or columns of themicroplate by aligning the fluorescence spectra from the referencestandard, (3) calibrating the optics to a known spectral wavelength (orwavelengths), (4) adjusting the multiple components of a light detectionsystem, such as lasers, beam splitters, neutral density filters, and/orthe like, (4) verifying instrument performance (alignment and event sizeand count) with specific sizes, and with specific numbers, of exemplaryfluorescent targets (particularly for cell counting devices), (5)locating grids within a microfluidic array device, and/or (6)correlating instrument response with the concentration of organic dye.Elaboration on these purposes, and/or additional purposes, are describedelsewhere herein.

The optical reference standard may have any properties suitable with itsfunction. These properties optionally can include, among others, (1) anability to withstand temperatures normally achieved in fluorescencedetection systems, such as real-time polymerase chain reaction (PCR)systems, such that it does not melt at temperatures below about 110° C.,(2) few and/or minor lot-to-lot variations in optical and physicalproperties, (3) high-intensity narrow-emission peaks (e.g., full widthat half-max 15 nm) at the wavelengths detected by the instrumentation,(4) uniform fluorescence emission spectra across all discrete wells, orpositions, or that are continuously uniform across the entire homogenoussurface, (5) stability for long periods of time (e.g., 5 years), withlittle or no degradation from photobleaching, and/or (6) low cost. Theemission peaks can be centered, in some embodiments, at about 520 nmcorresponding to FAM dye and at about 600 nm corresponding to ROX dye,when excited by a 488-nm wavelength light source, and/or in otherembodiments at the fluorescence wavelengths of Cy5 and Cy5.5 dyes, whenexcited by a red laser, and/or so on.

Instrument performance for cell-counting instruments can be validatedusing patterns of luminescent targets. Toward this end, a maskcontaining the desired shape, size, and quantity of fluorescence targetscan be applied over the fluorescence reference standard. Each target ismerely a hole through the mask material, which is opaque andnon-fluorescing, thereby providing a clear aperture of the desired shapeand size to the fluorescence reference standard material underneath. Themask can be an etched or machined chip. The mask can include severalwells that mimic the wells of normally used microplates (or other sampleholders), for example, a 1536-well SBS standard microplate. A particularpattern of targets can be contained within each well of the mask. Theparticular pattern in a well will depend on its function, that is,whether the standard is used for alignment, and/or to verify event sizeand count.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein.

1. A luminescence reference device comprising: a sample-support device, wherein the sample-support device is comprised of a plurality of fluid compartments; and at least one emissive element distributed in the plurality of fluid compartments of the sample-support device; wherein the at least one emissive element comprises at least one of a subtraction-based material or an addition-based material.
 2. The luminescence reference device of claim 1, wherein the sample-support device is a microtiter plate.
 3. The luminescence reference device of claim 1, wherein the sample-support device is a cell culture plate.
 4. The luminescence reference device of claim 1, wherein the at least one nanocrystalline material has an emission peak in the range between ultraviolet to near-infrared.
 5. The luminescence reference device of claim 1, wherein the at least one nanocrystalline material has an emission peak in the range between 500 nm to 700 nm.
 6. The luminescence reference device of claim 1, wherein the material comprises at least one of Ultem, photoluminescent-nanocrystal-containing material, quantum dots, or luminescent-glass-containing material.
 7. A system for biological reactions, comprising: a sample support configured to support one or more biological samples; a reference comprising: a light-producing element; at least one of a spectral filter layer, an intensify filter layer, or a protective coating layer; and a light source configured to illuminate the one or more biological samples; a detector to receive light from the one or more biological samples; an optical system configured to direct light from the reference to the detector; wherein the reference is configured to provide one or more of verification of system alignment or location of system grids.
 8. The system of claim 7, the light producing element is configured to produce one or more of emission, reflection, or scattering.
 9. The system of claim 7, further comprising a support layer configured to provide structural support for the mask.
 10. The system of claim 9, wherein the light producing element comprises a layer positioned on the support layer.
 11. The system of claim 9, wherein the light-producing element and the mask are separated by the at least one of a spectral filter layer, an intensify filter layer, and a protective coating layer.
 12. The system of claim 7, further comprising a light-blocking element bounding the light-producing element.
 13. The system of claim 12, wherein the light-blocking element forms a mask.
 14. The system of claim 7, further comprising an insulator configured to thermally insulate the reference
 15. The system of claim 14, wherein the insulator is coupled to the light-blocking elements.
 16. The system of claim 14, wherein the insulator is configured for use in a thermal cycler.
 17. The system of claim 14, wherein the insulator is configured for use in a thermal cycler having a heated lid.
 18. A method of verifying alignment of a system for biological reactions, comprising: providing a sample support configured to support one or more biological samples; providing a reference comprising: a light-producing element; at least one of a spectral filter layer, an intensify filter layer, or a protective coating layer; and illuminating the one or more biological samples with a light source; receiving light from the one or more biological samples at a detector; directing light from the reference to the detector using an optical system; based at least in part on the light directed from the reference to the detector, verifying system alignment. 